Endothelial & neuronal NO synthase enzymes (eNOS & nNOS) produce NO in a Ca2+/calmodulin (CaM) dependent manner. CaM-activated NO synthesis requires a large conformational change, in which the flavin mononucleotide (FMN) domain shuttles between NOS's electron-accepting input state and electron-donating output state to deliver electrons across the domains of the protein. In the output state, the FMN and heme domains form a docking complex, thus enabling interdomain electron transfer (IET) between the FMN and the catalytic heme center. Recent mass spectrometry and cryo-electron microscopy studies have provided a general view of the conformational changes and domain placement during the IET and NOS catalysis. Yet, there is still much unknown about how CaM activates the rate-limiting FMN/heme IET in the NOS output state. Research from our lab demonstrates that: (i) CaM controls formation of the output state by facilitating interdomain FMN/heme interactions; (ii) a CaM-responsive autoregulatory insert in the nNOS FMN domain stabilizes the output state; and (iii) the observed IET rate is limited by the relatively infrequen formation of the IET-competent docking complex. Despite significant progress, the roles of specific residues at the domain docking interfaces in determining the FMN/heme IET kinetics & conformation and population of the docked FMN/heme complex remain largely unclear. Our Aims address this gap through combined approaches of laser flash photolysis, pulsed electron paramagnetic resonance (EPR), molecular dynamics, and molecular biology, to achieve a molecular-level understanding of CaM-activation of the NOS output state. Based on recent results, the overall hypothesis is that specific NOS/CaM interactions and intrinsic NOS control elements synergistically regulate NOS function by facilitating the FMN/heme interdomain docking. In Aim 1, we will identify specific NOS and CaM interaction sites by focusing on mutations in the NOS heme domain and CaM surface residues at the predicted CaM/heme domain interface. We will use IET kinetics and pulsed EPR to measure changes resulting from specific mutations. This will reveal the roles of specific residues in facilitating the FMN heme IE and in forming the interfaces between the heme domain, FMN domain, and CaM. Aim 2 will determine how CaM-responsive control elements regulate NOS function by focusing on eNOS phosphorylations at the sites of potential importance for stroke intervention. We will analyze phosphomimetic mutants coupled with in vitro phosphorylated eNOS to define mechanistic roles of the phosphorylations in eNOS regulation. The proposed studies will provide significant insights into the molecular underpinnings of CaM-controlled formation of the NOS output state for NO production, and may help rational development of new selective NOS modulators targeting the domain docking interfaces related to the electron transfer.